Increasing demands for materials and fabrics that are both lightweight and flexible without compromising strength and durability has created a need for new fibers possessing higher tolerances for such properties as elasticity, denier, tensile strength and modulus. The search for a better fiber has led to the investigation of fibers produced in nature, some of which possess remarkable qualities. One of those fibers is derived from spider or insect silk, which includes a group of externally spun fibrous protein secretions.
Silks are produced by over 30,000 species of spiders and by many other insects, particularly in the order Lepidoptera. Few of these silks have been studied in detail. The cocoon silk of the domesticated silkworm Bombyx mori and the dragline silk of the orb-weaving spider Nephila clavipes are among the best characterized. Although the structural proteins from the cocoon silk and the dragline silk are quite different from each other in their primary amino acid sequences, they share remarkable similarities in many aspects. They are extremely glycine and alanine-rich proteins. Fibroin, a structural protein of the cocoon silk, contains 42.9% glycine and 30% alanine. Spidroin 1, a major component of the dragline silk, contains 37.1% glycine and 21.1% alanine. They are also highly repetitive proteins. The conserved crystalline domains in the heavy chain of the Fibroin and a stretch of polyalanine in Spidroin 1 are repeated numerous times throughout entire molecules. These crystalline domains are surrounded by larger non-repetitive amorphous domains in every 1 to 2 kilobases in the heavy chain of Fibroin, and by shorter repeated GXG amorphous domains in tandem in Spidroin 1. They are also shear sensitive due to their high copy number of the crystalline domains. During fiber spinning, the crystalline repeats are able to form anti-parallel-pleated sheets, so that silk protein is turned into semi-crystalline fiber with amorphous flexible chains reinforced by strong and stiff crystals.
Spider dragline silk has a tensile strength of over 200 ksi with an elasticity of nearly 35%, which makes it more difficult to break than either KEVLAR™ fibers or steel. When spun into fibers, spider silk may have application in the bulk clothing industries as well as being applicable for certain kinds of high strength uses such as rope, surgical sutures, flexible tie downs for certain electrical components and even as a biomaterial for implantation (e.g., artificial ligaments or aortic banding). Additionally these fibers may be mixed with various plastics and/or resins to prepare a fiber-reinforced plastic and/or resin product.
Traditional silk production from silkworm involves growing mulberry leaves, raising silkworms, harvesting cocoons, and processing of silk fibers. It is labor intensive and time consuming and therefore prohibitively expensive. The natural defects of the silkworm silk, such as the tendency to wrinkle and the irregularity of fiber diameter further limits its application. Similarly, the mass production of the dragline silk from spiders is not plausible because only small amounts are available from each spider. Furthermore, multiple forms of spider silks are produced simultaneously by any given spider. The resulting mixture has less application than a single isolated silk because the different spider silk proteins have different properties and are not easily separated. Thus, the prospect of producing commercial quantities of spider silk from a natural source is not a practical one and there remains a need for an alternate mode of production.
By using molecular recombination techniques, one can introduce foreign genes or artificially synthesized DNA fragments into different host organisms for the purpose of expressing desired protein products in commercially useful quantities. Such methods usually involve joining appropriate fragments of DNA to a vector molecule, which is then introduced into a recipient organism by transformation. Transformants are selected using a selectable marker on the vector, or by a genetic or biochemical screen to identify the cloned fragment.
While the techniques of foreign gene expression in the host cell are well known and widely practiced, the synthesis of foreign polypeptides containing high numbers of repeating units poses unique problems. Genes encoding proteins of this type are prone to genetic instability due to the repeating sequences, which result in truncated product instead of the full size protein.
The recent advances in cDNA sequencing of cocoon silk and dragline silk have permitted the synthesis of artificial genes for spider silk-like proteins with sequence and structural similarity to the native proteins. These artificial genes mimicked sequence arrays of natural cocoon silk from B. mori and dragline silk from N. clavipes, and had been introduced into microorganisms such as Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae. Synthetic spider silk proteins have been produced in these microorganisms through fermentation.
Many recombinant proteins have been produced in transgenic plants. Plant genetic engineering combines modern molecular recombination technology and agricultural crop production. However there are striking compositional and structural differences between silks and spider silk-like proteins and native plant proteins. For example, spider silk-like proteins are very glycine and alanine-rich, highly repetitive, and semi-crystalline in structure. These are characteristics not found in most plant proteins. Thus, introduction and expression of spider silk-like proteins genes in plant cells may pose a number of difficulties. For example, the repetitive sequence of spider silk-like protein genes may be a target for DNA deletion and rearrangement in plant cells.
Alternatively, translation of glycine and alanine-rich spider silk-like proteins might prematurely exhaust glycine and alanine and tRNA pools in plant cells. Finally, accumulation of semi-crystalline spider silk-like proteins may be recognized and degraded by the housekeeping mechanisms in the plant.
The methods known in the art for the expression of spider silk and spider silk-like proteins are useful for production in microbial systems. However, they are not applicable to the production of silk or spider silk-like proteins in plants. The use of a plant platform, such as maize cells for the production of silk and silk-like proteins, has several advantages over a microbial platform. For example, as a renewable resource, a plant platform requires far less energy and material consumption than microbial methods. Similarly, a plant platform represents a far greater available biomass for protein production than a microbial system.
There are several advantages of expressing spider silk proteins in plants over existing technologies. Corn endosperm, in particular, stores high concentrations of proteins in storage bodies, and targeting and processing can be directed by plant specific sequences.
The problem to be solved therefore is to provide a method to produce synthetic spider silk in the endosperm, leaf or shoot tissue of plants and to easily identify when synthetic spider silk proteins have been expressed in the plant endosperm, leaf or shoot tissue.
The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification.